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SMRT Sequencing Improves Reporting of CRISPR and Other Gene-Editing Outcomes

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The enthusiastic application of emerging gene-editing technologies such as CRISPR may run ahead of our ability to consider the efficiencies and outcomes of our genetic interventions. That is, genome editing procedures, at present, are hard to assess (and optimize), given the limitations of current reporting tools. These include gel-based assays, fluorescent reporters, and clone analysis. The shortcomings of these tools, which range from lack of sensitivity to the need to generate reporter cell lines, can be overcome with high-throughput sequencing of endogenous loci. This approach, however, presents its own problems. If confined to short read lengths, this approach cannot be used to analyze gene-editing frequencies when donor templates with long arms of homology are used. In many genome editing applications, donor DNA repair templates tend to be large.

To date, there has not been a sequencing platform capable of simultaneously assessing how frequently nonhomologous end-joining (NHEJ) occurs alongside targeted homologous repair (HR)—the main mechanisms of double-strand break repair. Now, however, researchers have shown that the long read lengths afforded by single molecule, real-time (SMRT) sequencing can provide a platform for simultaneously measuring genome editing outcomes by either mutagenic NHEJ or by HR at "any site of interest."

This advance was reported March 27 in Cell Reports, in an article entitled “Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing.” The article’s authors, who represent Stanford University, Georgia Institute of Technology, and Emory University, noted that their approach “can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs), RNA-guided endonucleases (CRISPR/Cas9), and zinc finger nucleases (ZFNs), and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred.”

Targeted genome editing with engineered nucleases, the authors observed, allows researchers to introduce precise sequence modifications at almost any site within the genome. In earlier work, the Stanford-led team cautioned that these genome modification strategies, which are initiated by a double-strand break created by an engineered nuclease, may not always produce the desired result: “The precise genome modification ... depends on whether the break is repaired in a mutagenic fashion by NHEJ or using a provided donor DNA molecule by (HR). If the break is repaired by NHEJ, small insertions or deletions are created at the site of the break. If the break is repaired by HR, however, defined sequence changes can be introduced at the site of the break, creating nucleotide specific modifications to the genome.”

In their Cell Reports article, the authors explained that SMRT DNA sequencing provides read lengths approaching 15 kb, allowing for analysis of gene-editing frequences when donor templates with long arms of homology are used. The use of such templates, the authors noted, is a common approach to increasing HDR efficiency in primary cells and for adding large gene inserts.

According to the authors, SMRT DNA editing offers the following advantages: “(1) sensitive measurement of genome editing in any cell type, including primary stem cells, without the need to make a stable reporter cell line, (2) measurement of modifications at endogenous loci regardless of transcriptional status, and (3) long sequencing read lengths that allow insight into a wide range of DNA repair outcomes when donor templates with long arms of homology are used.”

“Moreover, our strategy offers an approach for studying DNA repair pathway utilization when DNA breaks occur within genomic sites that have been difficult to study using previous methodologies,” the authors continued. “With the flexibility to evaluate engineered nucleases and targeting constructs directly at desired loci without the development of reporter systems, SMRT DNA sequencing can streamline the development of genome-editing projects and hasten the expansion of these technologies to a wider range of applications.”

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